JP2024511193A - Water-soluble polymer for preventing non-specific adsorption - Google Patents

Abstract

The present invention relates to a water-soluble copolymer that prevents non-specific adsorption of biological substances such as proteins. The water-soluble copolymer of the present invention is mainly used for reagents and particles used in clinical diagnosis, biochemical experiments, medical devices, and the like.

Description

Immunoassays exploit specific chemical interactions between antibodies and antigens to express unique proteomic "biomarker" signatures, useful for the discovery, detection, and monitoring of biological processes. . Immunoassays are gaining importance as both medical and research tools and in various scientific fields such as analytical chemistry, pharmacology, molecular cell biology, and clinical biochemistry. Immunoassays are now used in many laboratories around the world, and many companies are using them as diagnostic tools to quantify specific analytes such as pollutants, hormones, disease markers, and pathogens. We sell detection tools.

However, immunoassays suffer from the problem that the signal-to-noise ratio (SN ratio) is often low during the early stages of detection when the concentration level of the biomarker is relatively low. Highly sensitive tests are required, as low signal-to-noise ratios compromise assay reliability and lead to false-positive and false-negative results. This can be caused by high background noise due to non-specific adsorption of both target and non-target molecules to the assay platform. As a result, background noise becomes high and impedes improvement in sensitivity. Among various immunoassay techniques, chemiluminescence offers the highest detection limits compared to colorimetry and fluorescence. However, there is also room for improvement in the signal-to-noise ratio of chemiluminescent immunoassays.

In chemiluminescent immunoassays (CLIA), one potential source of high noise levels is conjugation with signal generators such as antibodies labeled with acridinium esters or isoluminol derivatives, alkaline phosphates, and horseradish peroxidase. This is due to non-specific adsorption of the secondary antibody. In the case of diagnosis in clinical samples, specific substances such as antigens are detected in coexisting biomolecules. Substances such as serum that coexist with biomolecules tend to be adsorbed onto the surface of the particles. This makes the surface of the particles more likely to non-specifically adsorb the secondary antibody labeled with the signal generator. As a result, noise becomes high, which hinders improvement of the S/N ratio.

A common approach to improving the signal-to-noise ratio of CLIA is to use biological materials such as albumin, casein, and gelatin as additives to prevent nonspecific adsorption. However, even after using these additives, non-specific adsorption remains. Furthermore, these biologically derived additives may have large batch-to-batch variations. For example, it may happen that some batches of albumin give good results while others have no effect at all. Since it is impossible to know which batches will be effective until they are tried, the burden of selection work increases. Furthermore, biological contamination can also be a problem when additives derived from living organisms are used. Furthermore, even if such additives prevent non-specific adsorption, cleaning with acids, alkalis, aqueous solutions of surfactants, etc. inhibits the effect and deteriorates durability. Attempts have been made to passivate surfaces using synthetic materials. Examples of synthetic polymers prepared to prevent nonspecific adsorption include polyoxyethylene (JP-A-5-103831, JP-A-2006-177914, and JP-A-2006-299045). Examples include copolymers having a phosphorylcholine group (JP-A-2006-176720 and JP-A-2006-258585). However, the effect of preventing nonspecific adsorption is not sufficient. This is because (1) the structure of copolymers is usually linear, so it may be difficult to cover a wide range using linear polymers, and (2) such block copolymers It is believed that the molecular weight of is typically low and it may be difficult to form thick coatings on the target surface. Other polymers include aminodextrans and modified dextran (EP2230515A1) of various molecular weights. Although these polymers may be able to prevent nonspecific adsorption to negatively charged proteins and biological materials, they are not as effective against positively charged proteins and biological materials. These polymers may not be effective against many biological samples, as biological samples are typically mixtures of antigens and antibodies, and may contain variously charged biomolecules within a single sample. There is. Thus, there is an unmet need for polymers that can prevent nonspecific adsorption to complex mixtures.

The present inventors have surprisingly discovered that nonspecific adsorption of biomolecules/biomaterials can be suppressed to a minimum by conjugating the polymer defined herein to the surface of a substrate. Ta. This has the effect of minimizing background binding, improving the SN ratio of the assay, and enabling more accurate and reliable detection.

Aspects and embodiments of the invention are described in the numbered sections below.
1. A water-soluble crosslinked copolymer formed by crosslinking a first polymer chain to a second polymer chain containing a polycarboxylic acid,
The first polymer chain before crosslinking includes a structural unit (a) containing a zwitterionic phosphorylcholine group, a structural unit (b) containing an amine group, and a structural unit (c) containing a hydroxyl group,
The water-soluble crosslinked copolymer includes one or more crosslinks formed between a carboxylic acid group of the second polymer chain and an amine group of the first polymer chain;
Optionally, the amine group of the first polymer chain is a primary amine group.

2. The water-soluble crosslinked copolymer according to item 1,
The water-soluble crosslinked copolymer is
A first polymer chain containing a structural unit (A') and/or (A'') whose formula is shown below, a structural unit (B), and a structural unit (C),
A water-soluble crosslinked copolymer containing a second polymer chain containing the structural unit (D),
A water-soluble crosslinked copolymer comprising one or more crosslinks between the first polymer chain and the second polymer chain.
(In the formula,
X represents O or NH,
R1 represents a straight chain or branched alkylene group having 1 to 5 carbon atoms,
E represents a covalent bond, -C(O)O-, -OC(O)-, -NHC(O)-, or -C(O)NH-,
Rc represents a hydroxyl group-containing group,
Each R4 independently represents a methyl group or a hydrogen atom,
R6 represents a moiety containing an amine group (for example, a primary amine group), and R6b represents -COOH or -C(O)O-Y-COOH (Y represents a C _1-5 alkylene group; At least one R6 of the first polymer chain and R6b of the second polymer chain are covalently bonded via an amide bond formed between the amine group of R6 and the carboxylic acid of R6b) ,
each R7 is independently selected from the group consisting of a hydrogen atom, a methyl group, an ethyl group, an n-propyl group, an isopropyl group, a methoxyethyl group, a hydroxymethyl group, and a hydroxyethyl group;
R8 represents a carboxyl group or a hydrogen atom,
R9 is a covalent bond, a linear or branched alkylene having 1 to 10 carbon atoms, or 1 to 3 groups selected from the group consisting of methoxy, hydroxymethyl, hydroxyethyl, and methoxyethyl groups. represents a straight-chain alkylene having 1 to 5 carbon atoms, with substituents;
w, x, y, and z are each independently integers from 1 to 50. )

3. The water-soluble crosslinked copolymer according to item 1 or 2,
The water-soluble crosslinked copolymer has a weight average molecular weight of 2,000 to 1,000,000 Daltons, preferably 5,000 to 500,000 Daltons, and more preferably 10,000 to 250,000 Daltons.

4. The water-soluble crosslinked copolymer according to item 2 or 3,
A water-soluble crosslinked copolymer in which the first polymer chain is a random copolymer containing a structural unit (A') and/or (A''), a structural unit (B), and a structural unit (C). Polymer.

5. The water-soluble crosslinked copolymer according to any one of items 2 to 4,
the first polymer chain comprises a structural unit (A') (wherein X is O, R1 is methylene, and E is a covalent bond), or the first polymer chain comprises a structural unit A water-soluble crosslinked copolymer containing (A'').

6. The water-soluble crosslinked copolymer according to any one of items 2 to 5,
The structural unit (B) is allylamine hydrochloride, 4-vinylaniline, 2-aminoethyl methacrylate hydrochloride, 2-aminoethyl acrylate, aminoacrylate, N-(2-aminoethyl)methacrylamide hydrochloride, N- (3-aminopropyl)methacrylamide hydrochloride and one selected from the group consisting of 2-aminoethylphenothiazine methacrylate (for example, N-(2-aminoethyl)methacrylamide hydrochloride). A water-soluble crosslinked copolymer showing structural units.

7. The water-soluble crosslinked copolymer according to any one of items 2 to 5,
A water-soluble crosslinked copolymer in which the structural unit (B) has the following formula.
(In the formula, R3 represents O or NH, R4 is defined in the second term, and R6a is -Y-CH ₂ -NH ₂ or -Z-NH ₂ (In the formula, Y is C _1-5 alkylene group, Z represents C _6-12 aryl group))

8. The water-soluble crosslinked copolymer according to any one of items 2 to 7, wherein the structural unit (C) has the following formula.
(In the formula,
R2 represents a linear or branched alkylene group having 1 to 18 carbon atoms, a polyoxyalkylene group having 1 to 20 constitutional units, or an arylene group having 6 to 18 carbon atoms,
R3 represents O or NH,
R4 is defined in Section 2,
R5 is C _n H _2n−m+1 (OH) _m (in the formula, n represents an integer from 1 to 5, and m is 1, 2, or 3))

9. 9. The water-soluble crosslinked copolymer according to item 8, wherein m is 1.

10. The water-soluble crosslinked copolymer according to item 8 or 9,
A water-soluble crosslinked copolymer in which R2 represents ethylene glycol or propylene glycol having 1 to 20 repeating units.

11. The water-soluble crosslinked copolymer according to any one of items 2 to 7,
The structural unit (C) is 2-hydroxyethyl methacrylate, 2-hydroxyethyl acrylate, hydroxypolyethoxy (10) allyl ether, N-(2-hydroxypropyl) methacrylamide, glycerol monomethacrylate, 3-methacrylate Phenoxy-2-hydroxypropyl, hydroxypropyl methacrylate, 4-hydroxybutyl acrylate, 2-hydroxypropyl acrylate, N-hydroxyethylacrylamide, poly(ethylene glycol) methacrylate, poly(propylene glycol) methacrylate, and A water-soluble crosslinked copolymer which is a structural unit formed by polymerization of one selected from the group consisting of N-[tris(hydroxymethyl)methyl]acrylamide.

12. A water-soluble crosslinked copolymer according to any one of the preceding clauses, wherein the weight average molecular weight of the first polymer chain is from 500 to 100,000 Daltons, preferably from 2,000 to 50,000 Daltons. Copolymer.

13. The water-soluble crosslinked copolymer according to any one of items 2 to 12,
The structural unit (D) is poly(acrylic acid), polymethacrylic acid, polystyrene-block-poly(acrylic acid), polymaleic acid, poly(acrylic acid-co-maleic acid), poly(D,L-lactide-block) - acrylic acid), poly(acrylamide-co-acrylic acid), poly(N-isopropylacrylamide-co-acrylic acid), and poly(ethylene-co-acrylic acid). A water-soluble crosslinked copolymer that is a formed structural unit.

14. 14. The water-soluble crosslinked copolymer according to item 13, wherein the structural unit (D) is formed by polymerization of one selected from the group consisting of acrylic acid, methacrylic acid, and 2-carboxyethyl acrylate. A water-soluble crosslinked copolymer that is a structural unit.

15. A water-soluble cross-linked copolymer according to any one of the preceding clauses, wherein the weight average molecular weight of the second polymer chain is from 2,000 to 700,000 Daltons, preferably from 5,000 to 350,000 Daltons, more preferably from 10,000 Daltons. A water-soluble cross-linked copolymer that is 250,000 daltons.

16. The water-soluble crosslinked copolymer according to any one of the preceding clauses, wherein 5% to 75% of the carboxylic acid groups of the second polymer chain are crosslinked to the amine groups of the first polymer chain. and preferably 10% to 50% of the carboxylic acid groups of the second polymer chain are crosslinked to the amine groups of the first polymer chain.

17. A manufacturing method for preparing a water-soluble crosslinked copolymer according to any one of the preceding paragraphs, comprising:
(a) A monomer corresponding to structural unit (1), a monomer corresponding to structural unit (2), and a monomer corresponding to structural unit (3) are mixed in a solvent (e.g., water). The process of
(b1) a step of polymerizing the monomer mixture, optionally adding an initiator and heating to a temperature of 55° C. to 90° C. for 5 hours to 24 hours; ,
(c1) A step of conjugating the polymer obtained in step (b1) with a polycarboxylic acid to form a water-soluble crosslinked copolymer, optionally conjugating the polymer with a buffer having a pH of 4.5 to 8.5. A manufacturing method comprising a step of carrying out in the presence of.

18. 18. The manufacturing method according to item 17, further comprising the step of (d) conjugating the water-soluble crosslinked copolymer obtained in step (c1) to a base material.

19. The manufacturing method according to item 17,
(b2) A step of conjugating the polymer obtained in step (b1) to a base material before step (c1), or (c0) A step of conjugating the polycarboxylic acid of step (c1) to a base material before step (c1). A manufacturing method further comprising the step of conjugating the polymer obtained in 1 to a base material before conjugating the polymer to the polycarboxylic acid.

20. The manufacturing method according to item 18 or 19,
The substrate is selected from particles, hollow filters, plastic tubes, glass fibers, glass slides, microplates, and microfluidic devices (e.g., the substrate is selected from particles, hollow filters, microplates, and microfluidic devices). microfluidic devices), manufacturing methods.

21. The manufacturing method according to any one of items 18 to 20,
The material of the base material is polyurethane, polyacrylonitrile, polymethacrylate, carbon fiber, cellulose material, polyacrylamide, polyacrylate, polyolefin, poly(4-methylbutene), polystyrene, poly(ethylene terephthalate), polysiloxane, nylon, A method of manufacturing selected from polymer-based materials, organic materials, or composite materials, such as poly(vinyl butyrate), ferromagnetic materials, silica, or mixtures or composites of any of these.

22. According to any one of paragraphs 18 to 21, the substrate is functionalized with one or more selected from the group consisting of primary amines, epoxide groups, tosyl groups, and aldehyde groups. Manufacturing method described.

23. 23. The method of claim 22, wherein the substrate is functionalized with a primary amine.

24. A base material comprising the water-soluble crosslinked copolymer according to any one of Items 1 to 16.

Figure 1 shows the adsorption of proteins to various types of beads.

It has been surprisingly discovered that all or some of the above problems can be solved by using the water-soluble crosslinked copolymers described herein.

Biological samples are typically complex mixtures of lipids, antigens, antibodies, etc. Since these may be oppositely charged in the same environment, it is important that the surface is configured to inhibit non-specific adsorption of the complex mixture. For example, the isoelectric point of β-HCG is 6.2-6.6, and the isoelectric point of thyrotropin receptor (TSHR) is 8-10. This means that when the pH is neutral, β-HCG is positively charged and the thyrotropin receptor (TSHR) is negatively charged. To prevent non-specific adsorption of biological substances, ideally the surface should not adsorb either biological substance at the same buffer pH. Surprisingly, it has been found that conjugation of the polymers of the present invention to the surface of a substrate minimizes non-specific adsorption. Chemiluminescent immunoassays using this coating are believed to provide accurate and reliable detection as the signal-to-noise ratio is improved and background binding is minimized. Those skilled in the art will appreciate that the polymers of the present invention may also be useful for coating other materials where non-specific adsorption (eg, of biomolecules such as proteins) is desired.

In embodiments described herein, the term "comprising" refers to
It should be construed to include the configurations listed, but not to limit the existence of other configurations. The term "comprising" may also refer to instances where only the listed ingredients/components are intended to be present (e.g., the term "comprising" may refer to (Can also be replaced by the expression "consists of" or "consists essentially of"). It is expressly intended that both the broad and narrow interpretations be applicable to all aspects and embodiments of the invention. That is, the term "comprising" and its synonyms may be replaced by the expression "consists of" or "consists essentially of" or similar expressions; The reverse is also possible.

In the embodiments described herein, various features may be described in singular or plural form. In this specification, references in the singular are understood to include the plural, and references in the plural are understood to include the plural, unless such interpretation is technically illogical. It is expressly intended to be understood to include the singular.

The water-soluble crosslinked copolymer of the present invention is herein referred to as a polymer (of the present invention), a copolymer (of the present invention), a water-soluble copolymer (of the present invention), a water-soluble crosslinked copolymer (of the present invention), may be referred to as a polymer or other expression.

Some of the advantages of the water-soluble crosslinked copolymers and associated methods for achieving surfaces that prevent non-specific adsorption of biological substances on substrates include:
- The water-soluble crosslinked copolymer is conjugated to the surface of the substrate using only the most common bioconjugation reaction mechanisms. The conditions are mild and do not affect the composition of antibodies or ligands such as streptavidin.
- The water-soluble crosslinked copolymers are good substitutes for substances of biological origin such as albumin, casein, gelatin, etc. as additives. The use of water-soluble crosslinked copolymers as additives can reduce the risk of biological contamination.
- Unlike conventional methods in which additives such as albumin, casein, gelatin, etc. are attached to the substrate surface by physical adsorption, the present water-soluble crosslinked copolymer is covalently bonded to the surface. This configuration exhibits durability upon repeated washing with surfactant buffers.
- The water-soluble cross-linked copolymer exhibits excellent performance over a wide pH range and is resistant to proteins with various isoelectric points.
- The water-soluble cross-linked copolymer helps improve the signal-to-noise ratio and minimize background binding in chemiluminescent immunoassays, resulting in accurate and reliable detection and improved purification yields.

This water-soluble crosslinked copolymer is useful for preventing nonspecific adsorption of substances such as lipids, proteins, sugars, or nucleic acids, and cells. According to the present invention, the water-soluble crosslinked copolymer is a water-soluble crosslinked copolymer formed by crosslinking a first polymer chain to a second polymer chain containing a polycarboxylic acid,
The first polymer chain before crosslinking includes a structural unit (a) containing a zwitterionic phosphorylcholine group, a structural unit (b) containing an amine group (for example, a primary amine group), and a structural unit (c) containing a hydroxyl group. ) and,
The water-soluble crosslinked copolymer includes one or more crosslinks formed between the carboxylic acid group of the second polymer chain and the (primary) amine group of the first polymer chain, Good too.

The water-soluble crosslinked polymer of the present invention is
A first polymer chain containing a structural unit (A') and/or (A'') whose formula is shown below, a structural unit (B), and a structural unit (C),
a second polymer chain containing the structural unit (D),
The water-soluble crosslinked copolymer may include one or more crosslinks between the first polymer chain and the second polymer chain.
(In the formula,
X represents O or NH,
R1 represents a straight chain or branched alkylene group having 1 to 5 carbon atoms,
E represents a covalent bond, -C(O)O-, -OC(O)-, -NHC(O)-, or -C(O)NH-,
Rc represents a hydroxyl group-containing group,
Each R4 independently represents a methyl group or a hydrogen atom,
R6 represents a moiety containing an amine group (for example, a primary amine group), R6b represents -COOH or -C(O)O-Y-COOH (Y represents a C _1-5 alkylene group, and At least one R6 of the first polymer chain and R6b of the second polymer chain are covalently bonded via an amide bond formed between the (primary) amine group of R6 and the carboxylic acid of R6b. ing),
each R7 is independently selected from the group consisting of a hydrogen atom, a methyl group, an ethyl group, an n-propyl group, an isopropyl group, a methoxyethyl group, a hydroxymethyl group, and a hydroxyethyl group;
R8 represents a carboxyl group or a hydrogen atom,
R9 is a covalent bond, a linear or branched alkylene having 1 to 10 carbon atoms, or 1 to 3 groups selected from the group consisting of methoxy, hydroxymethyl, hydroxyethyl, and methoxyethyl groups. represents a straight-chain alkylene having 1 to 5 carbon atoms, with substituents;
w, x, y, and z are each independently integers from 1 to 50. )

The weight average molecular weight of the water-soluble crosslinked copolymer of the present invention is from 2,000 to 1,000,000 Daltons, preferably from 5,000 to 500,000 Daltons, and more preferably from 10,000 to 250,000 Daltons. If the weight average molecular weight of the water-soluble crosslinked copolymer is less than 2,000 Daltons, no significant effect of preventing non-specific adsorption of biological substances will be obtained. On the other hand, if the weight average molecular weight of the water-soluble crosslinked copolymer exceeds 1,000,000 Daltons, the polymer solution will thicken, making solution preparation impractical.

The first polymer chain of the water-soluble crosslinked copolymer is a random copolymer containing a structural unit (A') and/or (A''), a structural unit (B), and a structural unit (C). It may be a combination. For example, in some embodiments, the first polymer chain of the water-soluble crosslinked polymer is a random copolymer comprising a structural unit (A'), a structural unit (B), and a structural unit (C). It may be a combination. In some embodiments, the first polymer chain of the water-soluble crosslinked polymer is a random copolymer comprising a structural unit (A''), a structural unit (B), and a structural unit (C). It may be.

The proportion of carboxylic acid groups of the second polymer chain that are crosslinked to the amine groups of the first polymer chain may be from 5% to 75%, for example from 10% to 50%. good.

In some embodiments, the first polymer chain has a structural unit (A') where X is O, R1 is methylene, and E is a covalent bond. In some embodiments, the first polymer chain has a structural unit (A'').

In some embodiments, structural unit (B) is allylamine hydrochloride, 4-vinylaniline, 2-aminoethyl methacrylate hydrochloride, 2-aminoethyl acrylate, aminoacrylate, N-(2-aminoethyl) A structural unit formed by polymerizing one selected from the group consisting of methacrylamide hydrochloride, N-(3-aminopropyl)methacrylamide hydrochloride, and 2-aminoethylphenothiazine methacrylate.

In some embodiments, the structural unit (B) is allylamine hydrochloride, 2-aminoethyl methacrylate hydrochloride, 2-aminoethyl acrylate, aminoacrylate, N-(2-aminoethyl)methacrylamide hydrochloride, by polymerizing one selected from the group consisting of N-(3-aminopropyl)methacrylamide hydrochloride and 2-aminoethylphenothiazine methacrylate (for example, N-(2-aminoethyl)methacrylamide hydrochloride). The structural units formed are shown.

In some embodiments, the structural unit (B) is allylamine hydrochloride, 2-aminoethyl methacrylate hydrochloride, 2-aminoethyl acrylate, N-(2-aminoethyl)methacrylamide hydrochloride, and N A structural unit formed by polymerizing one selected from the group consisting of -(3-aminopropyl)methacrylamide hydrochloride.

In some embodiments, the structural unit (B) represents a structural unit formed by polymerizing allylamine hydrochloride.

In some embodiments, the structural unit (B) is a structural unit as defined above and may include a primary amine group.

In some embodiments, structural unit (B) has the formula:
(In the formula, R3 represents O or NH, R4 is as defined above, and R6a is -Y-CH ₂ -NH ₂ or -Z-NH ₂ (In the formula, Y is C _1-5 represents an alkylene group (for example, a C _1-3 alkylene group), and Z represents a C _6-12 aryl group).

In embodiments, R6a is -Y-CH ₂ -NH ₂ and R6a comprises a primary amine. In some embodiments, Y represents a C _1-3 alkylene group.

In some embodiments, structural unit (C) has the formula:
During the ceremony,
R2 represents a linear or branched alkylene group having 1 to 18 carbon atoms, a polyoxyalkylene group having 1 to 20 constitutional units, or an arylene group having 6 to 18 carbon atoms,
R3 represents O or NH,
R4 is as specified above,
R5 represents C _n H _2n−m+1 (OH) _m (wherein n represents an integer from 1 to 5, and m is 1, 2, or 3).

In some embodiments, R2 represents ethylene glycol or propylene glycol with 1-20 repeat units. In some embodiments, R2 represents a straight or branched alkylene group having 1 to 5 carbon atoms.

In some embodiments, R2 is a polyoxyalkylene group having 1 to 20 units, such as 3 to 10 units (eg, ethylene glycol or propylene glycol).

In some embodiments, m is 1. In some embodiments, n is 1, 2, or 3, such as 1 or 2.

In some embodiments, the building block (C) is
2-hydroxyethyl methacrylate, 2-hydroxyethyl acrylate, hydroxypolyethoxy (10) allyl ether, N-(2-hydroxypropyl) methacrylamide, monoglycerol methacrylate, 3-phenoxy-2-hydroxypropyl methacrylate, Hydroxypropyl methacrylate, 4-hydroxybutyl acrylate, 2-hydroxypropyl acrylate, N-hydroxyethylacrylamide, poly(ethylene glycol) methacrylate, poly(propylene glycol) methacrylate, and N-[tris(hydroxymethyl) ) methyl] acrylamide (eg, 2-hydroxyethyl methacrylate).

In some embodiments, structural unit (C) is one polymeric selected from the group consisting of hydroxypolyethoxy(10) allyl ether, poly(ethylene glycol) methacrylate, and poly(propylene glycol) methacrylate. It is a structural unit formed from

In some embodiments, the weight average molecular weight of said first polymer chain is from 500 to 100,000 Daltons, preferably from 2,000 to 50,000 Daltons.

In some embodiments, structural unit (D) is poly(acrylic acid), polymethacrylic acid, polystyrene-block-poly(acrylic acid), polymaleic acid, poly(acrylic acid-co-maleic acid), poly( D,L-lactide-block-acrylic acid), poly(acrylamide-co-acrylic acid), poly(N-isopropylacrylamide-co-acrylic acid), and poly(ethylene-co-acrylic acid) A structural unit formed from one selected polymerization, for example, a structural unit formed from one polymerization selected from the group consisting of acrylic acid, methacrylic acid, and 2-carboxyethyl acrylate.

In some embodiments, the weight average molecular weight of the second polymer chain is from 2,000 to 700,000 Daltons, preferably from 5,000 to 350,000 Daltons, and more preferably from 10,000 to 250,000 Daltons.

The present invention also provides a manufacturing method for preparing the water-soluble crosslinked copolymer of the present invention, comprising:
(a) A monomer corresponding to structural unit (1), a monomer corresponding to structural unit (2), and a monomer corresponding to structural unit (3) are mixed in a solvent (e.g., water). The process of
(b1) a step of polymerizing the monomer mixture, optionally adding an initiator and heating to a temperature of 55° C. to 90° C. for 5 hours to 24 hours; ,
(c1) A step of conjugating the polymer obtained in step (b1) with a polycarboxylic acid to form a water-soluble crosslinked copolymer, optionally conjugating the polymer with a buffer having a pH of 4.5 to 8.5. Provided is a manufacturing method comprising a step of performing the step in the presence of.

This method may further include a step (d) of conjugating the water-soluble crosslinked copolymer obtained in step (c1) to a base material.

Alternatively, immobilization on the substrate may be performed before completing the synthesis of the water-soluble crosslinked copolymer. For example, the method:
(b2) A step of conjugating the polymer obtained in step (b1) to a base material before step (c1), or (c0) A step of conjugating the polycarboxylic acid of step (c1) to a base material before step (c1). The structure may further include a step of conjugating the polymer obtained in 1 to a base material before conjugating the polymer to the polycarboxylic acid.

A more specific manufacturing method for preparing the copolymer of the present invention is shown below.
(a) mixing the monomer in a solvent such as water;
(b) initiating the polymerization by adding an initiator at a desired temperature, typically in the range of 55°C to 99°C;
(c) continuing the reaction under heating for 5 to 24 hours;
(d) a step of purifying the reactant by reprecipitation, extraction, dialysis, etc.;
(e) The polymer (terpolymer) obtained in step (d) is conjugated to the polyacid skeleton by a carbodiimide conjugation mechanism in a buffer (e.g., a buffer with a pH of 4.5 to 8.5). a step of causing
(f) a step of removing unreacted terpolymer through dialysis, a column, etc.
Optionally, the terpolymer may be conjugated to a polyacid backbone coated on a substrate, in which case step (f) is not necessary.

The substrate may be selected from particles, hollow filters, plastic tubes, glass fibers, glass slides, microplates, and microfluidic devices (for example, the substrate may be selected from particles, hollow filters, microfluidic devices). microfluidic devices, such as those selected from particles, hollow filters, and microfluidic devices).

The materials of the base material include polyurethane, polyacrylonitrile, polymethacrylate, carbon fiber, cellulose material, polyacrylamide, polyacrylate, polyolefin, poly(4-methylbutene), polystyrene, poly(ethylene terephthalate), polysiloxane, nylon, It may be selected from at least one of a polymer-based material, an organic material, or a composite material, such as poly(vinyl butyrate), a ferromagnetic material, silica, or a mixture or composite of any of these. .

Additionally, the substrate is functionalized with anchor groups selected from the group consisting of primary amines, epoxide groups, tosyl groups, and aldehyde groups, which can further react with the water-soluble copolymers of the present embodiments. You can leave it there.

Conjugation of the water-soluble branched copolymer of the present invention to a substrate is carried out by (1) dissolving the water-soluble branched copolymer in a buffer having a pH of 4.5 to 8.5, and (2) activating the water-soluble branched copolymer. (3) mixing the water-soluble branched copolymer after activation with the base material, and (4) washing and removing the unbound water-soluble branched copolymer. .

Optionally, the conjugation of the water-soluble copolymer of this embodiment to the substrate can be carried out by (1) dissolving the terpolymer in a buffer having a pH of 4.5 to 8.5, and (2) conjugating the surface of the substrate. (3) mixing the substrate with the terpolymer solution; and (4) washing and removing unbound water-soluble branched copolymer.

Optionally, the water-soluble branched copolymer of this embodiment is conjugated to the base material by (1) dissolving the water-soluble branched copolymer in a buffer having a pH of 4.5 to 8.5, and (2) conjugating the water-soluble branched copolymer with an epoxide group or a tosyl group. , and a base material having a functional group selected from aldehyde groups.

Materials and equipment:
Materials were purchased from the following sources:
Styrene: Tokyo Chemical Industry Co., Ltd. (TCI), stabilized with TBC (4-tert-butylcatechol), >99.0% (GC)
Azobisisobutyronitrile (AIBN): Sigma-Aldrich (acetone solution, 12wt%)
Sodium persulfate (Na ₂ S ₂ O ₈ , SPS): Alpha Acer, crystal, 98%
Polyvinylpyrrolidone (PVP, K30, MW=40,000): TCI, total nitrogen content 12.0% to 12.8% (anhydrous equivalent); maximum water content 7.0%, K value 26.0 to 34.0
Divinylbenzene (DVB, mixture of m and p forms): TCI, 50.0% (GC) (contains ethylvinylbenzene, diethylbenzene) (stabilized with 4-tert-butylcatechol)
2-Hydroxyethyl methacrylate: Sigma-Aldrich, ≧99.0%
2-carboxyethyl acrylate oligomer: Sigma-Aldrich, contains 2000ppm MEHQ as inhibitor
N-Hydroxysuccinimide (NHS), >98%, TCI
Ethanol: 99%, Aik Moh Acrylamide monomer (approximately 50% aqueous solution), TCI
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), >98%, TCI
Deionized (DI) water: obtained from ELGA Ultrapure Water Treatment Systems (PURELAB option)
10 phosphate buffered saline, Sigma-Aldrich
MES hydrate, >99.5%, Sigma-Aldrich Amine-PEG-carboxylic hydrochloride, Polyscience
2-(methacryloyloxy)ethyldimethyl-(3-sulfopropyl)ammonium hydroxide, Sigma-Aldrich
2-methacryloyloxyethylphosphorylcholine, Sigma-Aldrich Bovine serum albumin, Sigma-Aldrich Lysozyme, Sigma-Aldrich Stirrer: Wiggens, WB2000-M overhead stirrer Classic tube roller: SCILOGEX MX-T6-S

Mechanical stirring (200 rpm) was used in all polymerization processes. Before use, deionized water and ethanol were bubbled with nitrogen for 20 min to remove oxygen. All chemicals were used as received without purification. All polymerizations and reactions were performed under nitrogen protection using standard Schlenkline techniques.

Example 1
Synthesis of terpolymer In a 100 mL three-necked round bottom glass reactor equipped with a stirrer, add 50 g of water, 0.5 g of 2-methacryloyloxyethylphosphorylcholine as monomer (1), and 0.5 g of 2-methacryloyloxyethylphosphorylcholine as monomer (2). 0.01 g of N-(2-aminoethyl) methacrylamide hydrochloride and 1 g of 2-hydroxyethyl methacrylate as monomer (3) were added, and nitrogen was injected from one injection port to remove oxygen. After flushing with nitrogen for 10 minutes, the glass reactor was heated to 75°C. After the temperature of the glass reactor stabilized at 75° C., 5 mg of sodium persulfate was added as an initiator. The mixture was stirred for 24 hours to complete the polymerization. The obtained solution was purified by dialysis.

Example 2
Synthesis of polyacid skeleton In a 100 mL three-necked round bottom glass reactor equipped with a stirrer, 50 g of water and 1 g of 2-carboxyethyl acrylate as monomer (4) were added, and nitrogen was added through one inlet. The oxygen was removed by injection. After flushing with nitrogen for 10 minutes, the glass reactor was heated to 75°C. After the temperature of the glass reactor stabilized at 75° C., 5 mg of sodium persulfate was added as an initiator. The mixture was stirred for 24 hours to complete the polymerization. The obtained polycarboxylic acid solution was purified by dialysis using MES buffer.

Example 3
Preparation of water-soluble copolymers
EDC and NHS were dissolved separately in 25mM MES buffer to a concentration of 50mg/mL. EDC solution was added at a ratio of 20 μl to 1 mg of polycarboxylic acid skeleton, and then 20 μl of NHS solution was added. This solution was stirred for 30 minutes and then added to the terpolymer solution at a ratio of 25 mg of terpolymer to 1 mg of polycarboxylic acid. This mixture was stirred for an additional 2 hours. The obtained water-soluble copolymer solution was purified by dialysis.

Example 4
Conjugation of a water-soluble copolymer onto the surface of microspheres with primary amine moieties on the surface In a 250 mL three-necked round-bottomed glass reactor equipped with a stirrer, add AIBN solution (2 g, 12 wt% acetone solution) and purify with nitrogen. The acetone solvent was removed by injecting through one inlet. After flushing with nitrogen for 10 minutes, dry powdery AIBN was observed at the bottom of the reactor. Then PVP (0.2 g), ethanol (80 mL), and deionized water (20 mL) were added. The mixture was stirred at room temperature for 5 minutes to obtain a clear solution and the solution was heated to 60° C. in an oil bath before styrene (7.5 mL) was added. After 4 hours, it became a white colloidal solution, indicating that the styrene had polymerized. Then, a solution of DVB (3 mL DVB in 7 mL ethanol) was slowly added in a constant pressure addition funnel over 30 minutes. After the dropwise addition of the DVB solution was completed, the reaction (crosslinking polymerization) was continued for 3 hours. Then, a monomer solution of 2-hydroxyethyl methacrylate (0.75 g in 5 mL ethanol) and acrylamide monomer (0.75 g in 5 mL ethanol, neutralized with ammonia solution, 25% W/W) was added using a syringe. The reaction (polymerization) was continued for 6 hours to obtain a milky colloidal solution. The obtained beads are referred to as "A-1". The beads were centrifuged at 7000 G for 9 minutes and washed with ethanol. The supernatant was discarded and the microspheres were resuspended in 25mM MES buffer.

EDC and NHS were dissolved separately in 25mM MES buffer to a concentration of 50mg/mL. EDC solution was added at a ratio of 20 μl to 1 mg of water-soluble copolymer, and then 20 μl of NHS solution was added. This suspension was mixed with 3 mg of beads A-1 to give a concentration of 10 mg/ml. This mixture was stirred for an additional 2 hours. The resulting beads A-2 were washed with Tris buffer by centrifugation at 9000 rpm for 11 minutes. The supernatant was discarded and the microspheres were resuspended in 1PBS buffer.

Example 5
Conjugation of a terpolymer to the surface of microspheres with a polyacid backbone on the surface Conjugation of a water-soluble copolymer to the surface of microspheres with a polycarboxylic acid on the surface 250 mL three-necked round bottom with stirrer AIBN solution (2 g, 12 wt% acetone solution) was added to a glass reactor and nitrogen was injected through one inlet to remove the acetone solvent. After flushing with nitrogen for 10 minutes, dry powdery AIBN was observed at the bottom of the reactor. Then PVP (0.2 g), ethanol (80 mL), and deionized water (20 mL) were added. The mixture was stirred at room temperature for 5 minutes to obtain a clear solution and the solution was heated to 60° C. in an oil bath before styrene (7.5 mL) was added. After 4 hours, it became a white colloidal solution, indicating that the styrene had polymerized. Then, a solution of DVB (3 mL DVB in 7 mL ethanol) was slowly added in a constant pressure addition funnel over 30 minutes. After the dropwise addition of the DVB solution was completed, the reaction (crosslinking polymerization) was continued for 3 hours. Then, a monomer solution of 2-hydroxyethyl methacrylate (0.75 g in 5 mL ethanol) and carboxyethyl acrylate oligomer (0.75 g in 5 mL ethanol, neutralized with ammonia solution, 25% W/W ) was added using a syringe. The reaction (polymerization) was continued for 6 hours to obtain a milky colloidal solution. The obtained beads are referred to as "B-1". The beads were washed with ethanol for 9 minutes using centrifugation at 7000G centrifugal force. The supernatant was discarded and the microspheres were resuspended in 25mM MES buffer.

EDC and NHS were dissolved separately in 25mM MES buffer to a concentration of 50mg/mL. EDC solution was added at a ratio of 20 μl to 3 mg of beads B-1, and then 20 μl of NHS solution was added. This mixture was stirred for 30 minutes. The resulting activated beads were centrifuged at 7000 G centrifugal force for 9 minutes to remove the supernatant. Thereafter, the terpolymer solution (20 mg/ml) was added to the activated beads and stirred for 2 hours. The resulting beads B-2 were washed with Tris buffer for 11 minutes using centrifugation at 9000 rpm. The supernatant was discarded and the microspheres were resuspended in 1PBS buffer.

Example 6
Comparative example 6-1
Water-soluble copolymer (II) was synthesized by performing the steps in Examples 1, 2, and 3. However, monomer (1) was changed to 2-(methacryloyloxy)ethyldimethyl-(3-sulfopropyl)ammonium hydroxide. Microsphere sample C-1 was prepared by following each step of Example 4.

Comparative example 6-2
Water-soluble copolymer (III) was synthesized by performing each of the above steps. However, monomer (3) was not added during the terpolymer synthesis in Step 1. Microsphere sample C-2 was prepared by following the steps of Example 4.

Comparative example 6-3
Microsphere sample B-1 was conjugated with amine-PEG-carboxylic hydrochloride (Mn 2000 Daltons) using a protocol similar to Example 5. EDC and NHS were dissolved separately in 25mM MES buffer to a concentration of 50mg/mL. EDC solution was added at a ratio of 20 μl to 3 mg of beads B-1, and then 20 μl of NHS solution was added. This mixture was stirred for 30 minutes. The resulting activated beads were centrifuged at 9000 rpm for 11 minutes to remove the supernatant. Thereafter, the terpolymer solution (20 mg/ml) was added to the activated beads and stirred for 2 hours. The resulting beads C-3 were centrifuged at 9000G for 11 minutes and washed with Tris buffer. The supernatant was discarded and the microspheres were resuspended in 1PBS buffer.

Example 7
Non-specific adsorption test
BSA and lysozyme have different isoelectric points. BSA (isoelectric point 5.5) and lysozyme (isoelectric point 11) are commonly used in research as model proteins. In this evaluation, both were used to evaluate the nonspecific adsorption effect of different types of bead surfaces.

BSA and lysozyme were each dissolved in 1 PBS buffer solution to prepare stock solutions with a concentration of 0.2 mg/ml, respectively. Protein was added at a total dry mass of 0.1 mg per mg dry mass of bead sample (10 mg/ml, 0.1 ml). Each bead sample prepared in Examples 4, 5, and 6 was tested.

The bead-protein suspension was stirred at 25°C for 2 hours to obtain adsorption equilibrium. Samples were centrifuged for 11 minutes at 9000 rpm to sediment particles. The protein content of the supernatant was determined by UV-Vis spectroscopy at 280 nm. The amount of protein adsorbed on the bead surface was calculated using equation (1).
where y _ad indicates the percentage (%) of protein adsorbed on the bead surface;
P _tot indicates the total amount of protein added (mg),
P _s indicates the amount of protein in the supernatant (mg).

As shown in FIG. 1, when the water-soluble copolymer of this embodiment is coated on the bead surface (see A-2 and B-2), no adsorption of either BSA or lysozyme is observed. When only the polyacid backbone was coated on the bead surface (see B1), the adsorption of lysozyme was significantly higher than that of BSA. This is thought to be because BSA is negatively charged in the 1PBS buffer, while lysozyme is positively charged and is easily adsorbed to negatively charged surfaces. The combination of 2-methacryloyloxyethylphosphorylcholine and 2-hydroxyethyl methacrylate was able to prevent the adsorption of both BSA and lysozyme. This result was better than 2-(methacryloyloxy)ethyldimethyl-(3-sulfopropyl)ammonium hydroxide alone or 2-methacryloyloxyethylphosphorylcholine alone. Also, linear PEG, an antifouling brush commonly used to prevent nonspecific adsorption, showed the highest nonspecific adsorption (i.e., had the worst results) for both proteins.

It is therefore clear that the present invention provides an improved coating for preventing non-specific adsorption of biomolecules to surfaces. The present invention can prevent non-specific adsorption to both positively charged surfaces (amines, Example 4) and negatively charged surfaces (carboxylic acids, Example 5).

Claims (24)

A water-soluble crosslinked copolymer formed by crosslinking a first polymer chain to a second polymer chain containing a polycarboxylic acid,
The first polymer chain before crosslinking includes a structural unit (a) containing a zwitterionic phosphorylcholine group, a structural unit (b) containing an amine group, and a structural unit (c) containing a hydroxyl group,
The water-soluble crosslinked copolymer includes one or more crosslinks formed between a carboxylic acid group of the second polymer chain and an amine group of the first polymer chain;
Optionally, the water-soluble crosslinked copolymer, wherein the amine group of the first polymer chain is a primary amine group. The water-soluble crosslinked copolymer according to claim 1,
The water-soluble crosslinked copolymer is
A first polymer chain containing a structural unit (A') and/or (A'') whose formula is shown below, a structural unit (B), and a structural unit (C),
A water-soluble crosslinked copolymer containing a second polymer chain containing the structural unit (D),
A water-soluble crosslinked copolymer comprising one or more crosslinks between the first polymer chain and the second polymer chain.
(In the formula,
X represents O or NH,
R1 represents a straight chain or branched alkylene group having 1 to 5 carbon atoms,
E represents a covalent bond, -C(O)O-, -OC(O)-, -NHC(O)-, or -C(O)NH-,
Rc represents a hydroxyl group-containing group,
Each R4 independently represents a methyl group or a hydrogen atom,
R6 represents a moiety containing an amine group (for example, a primary amine group), and R6b represents -COOH or -C(O)O-Y-COOH (Y represents a C _1-5 alkylene group; At least one R6 of the first polymer chain and R6b of the second polymer chain are covalently bonded via an amide bond formed between the amine group of R6 and the carboxylic acid of R6b) ,
each R7 is independently selected from the group consisting of a hydrogen atom, a methyl group, an ethyl group, an n-propyl group, an isopropyl group, a methoxyethyl group, a hydroxymethyl group, and a hydroxyethyl group;
R8 represents a carboxyl group or a hydrogen atom,
R9 is a covalent bond, a linear or branched alkylene having 1 to 10 carbon atoms, or 1 to 3 groups selected from the group consisting of methoxy, hydroxymethyl, hydroxyethyl, and methoxyethyl groups. represents a straight-chain alkylene having 1 to 5 carbon atoms, with substituents;
w, x, y, and z are each independently integers from 1 to 50. ) The water-soluble crosslinked copolymer according to claim 1 or 2,
The water-soluble crosslinked copolymer has a weight average molecular weight of 2,000 to 1,000,000 Daltons, preferably 5,000 to 500,000 Daltons, and more preferably 10,000 to 250,000 Daltons. The water-soluble crosslinked copolymer according to claim 2 or 3,
A water-soluble crosslinked copolymer in which the first polymer chain is a random copolymer containing a structural unit (A') and/or (A''), a structural unit (B), and a structural unit (C). Polymer. The water-soluble crosslinked copolymer according to any one of claims 2 to 4,
the first polymer chain comprises a structural unit (A') (wherein X is O, R1 is methylene, and E is a covalent bond), or the first polymer chain comprises a structural unit A water-soluble crosslinked copolymer containing (A''). The water-soluble crosslinked copolymer according to any one of claims 2 to 5,
The structural unit (B) is allylamine hydrochloride, 4-vinylaniline, 2-aminoethyl methacrylate hydrochloride, 2-aminoethyl acrylate, aminoacrylate, N-(2-aminoethyl)methacrylamide hydrochloride, N- (3-aminopropyl)methacrylamide hydrochloride and one selected from the group consisting of 2-aminoethylphenothiazine methacrylate (for example, N-(2-aminoethyl)methacrylamide hydrochloride). A water-soluble crosslinked copolymer showing structural units. The water-soluble crosslinked copolymer according to any one of claims 2 to 5,
A water-soluble crosslinked copolymer in which the structural unit (B) has the following formula.
(In the formula, R3 represents O or NH, R4 is as defined in claim 2, and R6a is -Y-CH ₂ -NH ₂ or -Z-NH ₂ (In the formula, Y is C _1-5 alkylene group, Z represents C _6-12 aryl group)) The water-soluble crosslinked copolymer according to any one of claims 2 to 7, wherein the structural unit (C) has the following formula.
(In the formula,
R2 represents a linear or branched alkylene group having 1 to 18 carbon atoms, a polyoxyalkylene group having 1 to 20 constitutional units, or an arylene group having 6 to 18 carbon atoms,
R3 represents O or NH,
R4 is defined in claim 2,
R5 is C _n H _2n−m+1 (OH) _m (in the formula, n represents an integer from 1 to 5, and m is 1, 2, or 3)) The water-soluble crosslinked copolymer according to claim 8, wherein m is 1. The water-soluble crosslinked copolymer according to claim 8 or 9, wherein R2 represents ethylene glycol or propylene glycol having 1 to 20 repeating units. The water-soluble crosslinked copolymer according to any one of claims 2 to 7,
The structural unit (C) is 2-hydroxyethyl methacrylate, 2-hydroxyethyl acrylate, hydroxypolyethoxy (10) allyl ether, N-(2-hydroxypropyl) methacrylamide, glycerol monomethacrylate, 3-methacrylate Phenoxy-2-hydroxypropyl, hydroxypropyl methacrylate, 4-hydroxybutyl acrylate, 2-hydroxypropyl acrylate, N-hydroxyethylacrylamide, poly(ethylene glycol) methacrylate, poly(propylene glycol) methacrylate, and A water-soluble crosslinked copolymer which is a structural unit formed from the polymerization of one selected from the group consisting of N-[tris(hydroxymethyl)methyl]acrylamide (for example, 2-hydroxyethyl methacrylate). A water-soluble crosslinked copolymer according to any one of the preceding claims, wherein the weight average molecular weight of the first polymer chain is from 500 to 100,000 Daltons, preferably from 2,000 to 50,000 Daltons. Crosslinked copolymer. The water-soluble crosslinked copolymer according to any one of claims 2 to 12,
The structural unit (D) is poly(acrylic acid), polymethacrylic acid, polystyrene-block-poly(acrylic acid), polymaleic acid, poly(acrylic acid-co-maleic acid), poly(D,L-lactide-block) - acrylic acid), poly(acrylamide-co-acrylic acid), poly(N-isopropylacrylamide-co-acrylic acid), and poly(ethylene-co-acrylic acid). A water-soluble crosslinked copolymer that is a formed structural unit. The water-soluble crosslinked copolymer according to claim 13,
A water-soluble crosslinked copolymer, wherein the structural unit (D) is a structural unit formed by polymerization of one selected from the group consisting of acrylic acid, methacrylic acid, and 2-carboxyethyl acrylate. A water-soluble crosslinked copolymer according to any one of the preceding claims, comprising:
A water-soluble crosslinked copolymer, wherein the weight average molecular weight of the second polymer chain is from 2,000 to 700,000 Daltons, preferably from 5,000 to 350,000 Daltons, more preferably from 10,000 to 250,000 Daltons. A water-soluble crosslinked copolymer according to any one of the preceding claims, wherein from 5% to 75% of the carboxylic acid groups of the second polymer chain are crosslinked to the amine groups of the first polymer chain. and preferably, 10% to 50% of the carboxylic acid groups of the second polymer chain are crosslinked to the amine groups of the first polymer chain. A manufacturing method for preparing a water-soluble crosslinked copolymer according to any one of the preceding claims, comprising:
(a) A monomer corresponding to structural unit (1), a monomer corresponding to structural unit (2), and a monomer corresponding to structural unit (3) are mixed in a solvent (e.g., water). The process of
(b1) a step of polymerizing the monomer mixture, optionally adding an initiator and heating to a temperature of 55° C. to 90° C. for 5 hours to 24 hours; ,
(c1) A step of conjugating the polymer obtained in step (b1) with a polycarboxylic acid to form a water-soluble crosslinked copolymer, optionally conjugating the polymer with a buffer having a pH of 4.5 to 8.5. A manufacturing method comprising a step of carrying out in the presence of. The manufacturing method according to claim 17,
(d) A manufacturing method further comprising the step of conjugating the water-soluble crosslinked copolymer obtained in step (c1) to a base material. The manufacturing method according to claim 17,
(b2) A step of conjugating the polymer obtained in step (b1) to a base material before step (c1), or (c0) A step of conjugating the polycarboxylic acid of step (c1) to a base material before step (c1). A manufacturing method further comprising the step of conjugating the polymer obtained in the above to a base material before conjugating the polymer to the polycarboxylic acid. The manufacturing method according to claim 18 or 19,
The substrate is selected from particles, hollow filters, plastic tubes, glass fibers, glass slides, microplates, and microfluidic devices (e.g., the substrate is selected from particles, hollow filters, microplates, and microfluidic devices). microfluidic devices), manufacturing methods. The manufacturing method according to any one of claims 18 to 20,
The material of the base material is polyurethane, polyacrylonitrile, polymethacrylate, carbon fiber, cellulose material, polyacrylamide, polyacrylate, polyolefin, poly(4-methylbutene), polystyrene, poly(ethylene terephthalate), polysiloxane, nylon, A method of manufacturing selected from polymer-based materials, organic materials, or composite materials, such as poly(vinyl butyrate), ferromagnetic materials, silica, or mixtures or composites of any of these. The manufacturing method according to any one of claims 18 to 21,
The manufacturing method, wherein the substrate is functionalized with one or more selected from the group consisting of primary amines, epoxide groups, tosyl groups, and aldehyde groups. 23. The method of claim 22, wherein the substrate is functionalized with a primary amine. A base material comprising the water-soluble crosslinked copolymer according to any one of claims 1 to 16.